A combined method for establishing keratinocyte cultures from cervical biopsy specimens and creating patent-derived 3-dimensional organotypic raft cultures

Abstract

High-risk types of human papillomavirus (HPV) are responsible for nearly all instances of cervical cancer, and a significant proportion of head and neck cancers. Studying HPV in the laboratory requires the cultivation of host keratinocytes to facilitate the viral lifecycle. In addition, HPV will not completely undergo its lifecycle in monolayer cell culture, but requires the differentiation process of keratinocytes. This can be accomplished by employing organotypic raft cultures, which simulates full-thickness skin in vitro. Though this method can reproduce the viral lifecycle, there are still numerous hurdles when modelling disease. In the first phase of this study, we utilize published methods to culture cervical keratinocytes from 25 different cervical lesion biopsies, establishing cultures from 8 (~32%). Biopsy specimens were digested with collagenase I and grown in keratinocyte growth medium (KGM) containing 5% fetal bovine serum for 4 days, after which, cultures were switched to serum-free KGM to avoid fibroblast overgrowth. The median in vitro lifespan of cultures was 4 weeks, which typically yielded less than a confluent T-75 of cells. In the second phase of this study, the media was switched from KGM to EpiLife, which has more organic components, and contains insulin-like growth factor I in place of insulin, which has more mitogenic potential and increases keratinocyte lifespan in vitro. Biopsies were processed under the same conditions, and after removing the media containing 5% serum, the Rho-associated kinase (ROCK) inhibitor Y-27632 was added to EpiLife media to further increase culture lifespan. Cultures were established from 6 of 14 (~42%) newly acquired biopsies, 4 of which survived for several passages (range 2-7) across T-75 flasks (flasks seeded at 10% confluence and passaged when >80% confluent). The improved culture conditions effectively halved the doubling time, but maintained approximately the same culture lifespan (median 4.5 weeks). Organotypic raft cultures were prepared using 5 different patient samples; most had differentiated at the time of 3D culturing, while others yielded only a few cell layers upon cross sectioning and H&E staining. Future work will improve upon our culture media formulations to facilitate stratification of raft cultures, and potentially improve culture lifespan further.